The present invention relates to the isolation of Jatropha curcas curcin genes and tissue-specific promoters and to the production of curcin-deficient Jatropha plants. More specifically, the present invention relates to the isolation of Jatropha curcas Curcin 1, Curcin 2 and Curcin 2A. The present invention further relates to of the Curcin 1, Curcin 2A and Curcin 2 genes and more particularly to tissue specific promoters of the Curcin 1 and Curcin 2A genes. The present invention further relates to production of curcin-deficient transgenic jatropha plants by using RNAi technology to suppress curcin gene expression.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the Bibliography.
Tissue specific promoters are strongly desired for plant biotechnology to express gene-of-interest in the particular plant tissue at right time (Mansoor et al., 2006). Plant seed endosperm accumulates storage materials, such as starch, proteins and lipids (Berger et al., 2006; Hannah and James, 2008). The endosperm-specific promoters have been used to drive the expression of genes that are involved in biosynthesis pathways of these storage materials (Roesler et al., 1997; Plant et al., 1994; Kuwano et al., 2009). The insecticidal toxins, such as Bacillus thuringiensis (Bt) δ-endotoxins, have been used to control insects (Christou et al., 2006; Roh et al., 2007). Although Bt toxin has high specificity as an insecticidal toxin and safety for the environment, it is still desirable to have Bt toxin specifically expressed in leaf tissues rather than in seeds and fruits (Datta et al., 1998). Therefore, there is an increasing demand of specific promoter to control the expression of gene of interest in specific tissues at particular developmental stages. In addition, the multi-gene transformation system was used for delivery several genes simultaneously constructed in one expression vector (Lin et al., 2003; Chen et al., 2006; Wakasa et al., 2006). Each of these multiple genes needs a different promoter to drive them to avoid the gene silencing. However, a lack of suitable promoters is a critical limiting factor for such research (Qu et al., 2008).
Jatropha (Jatropha curcas) is a small tropical, woody plant belonging to the Euphorbiaceae family. It is a dicotyledonous plant and its seeds contain as much as 40% oil (Bringi, 1987). The oil from jatropha could be an efficient substitute for fossil fuel (Augustus et al., 2002; Azam et al., 2005; Forson et al., 2004; Pramanik, 2003). However, Jatropha suffers from several shortcomings that may limit its wide adoption. The productivity of the plant is constrained by the unfavourable male to female flower ratio and its oil content has not been optimized by breeding. This plant is also sensitive to biotic stresses such as viral (Narayanna et al., 2007), fungal and bacterium pathogens and abiotic stresses, especially cold and drought (http colon www dot jatropha dot orgy. The presence of several toxic components (e.g. the protein toxin, curcin, and the cancer-causing agent phorbol esters) in seeds and leaves of the plant possess health hazards for farmers and bioprocess workers in the Jatropha industry.
Curcin is a toxin protein identified in jatropha seeds (Stirpe et al. 1976). The presence of curcin as well as other toxins in jatropha seeds prevents the use of jatropha seed meals as animal feed (Makkar et al., 1997). Curcin belongs to the type I of ribosome inactivating proteins (RIPS) that has RNA N-glycosidase activity and can irreversibly inactivate ribosomes (Barbieri, 1993, Lin et al., 2003a). Currently, five curcin proteins have been deposited to GenBank and their accession numbers are AAL58089 (Lin et al., 2003a), AAL86778 (Lin et al., 2003b), ABZ04128, AAR08395 and ABW17545. These curcin proteins can be divided into two types based on the length of their amino acid residues. The precursors of type 1 curcin proteins consist of 293-aa residues while the precursors of type 2 curcin proteins contain 309-aa residues. Lin et al. (2003a) identified a type 1 curcin protein from jatropha seeds that encodes a 32-kDa curcin precursor with a 42-amino acid signal peptide. Wei et al. (2005) cloned a type 2 curcin gene. The curcin gene, designated as Curcin 2, was found to be induced by stress. Members of curcin proteins within a type display 93% to 98% identity in amino acids while members between two different types show 87%.
An important strategy to improve agronomic and quality traits of Jatropha curcas is by genetic modification. Transgenic Jatropha plants can be generated expressing homologous or heterologous gene sequences. In many instances, over-expression or silencing by RNA interference (RNAi) of one or more homologous genes of defined function is desired. Gene sequences of Jatropha can be obtained from cDNA and genomic libraries and functions of genes can be tentatively assigned by sequence homology with other plant genes of known function. Tissue-specific promoters are often utilized to express homologous or heterologous gene sequences in the desired tissues.
RNAi is a post-transcriptional, targeted gene-silencing technique that uses double-stranded RNA (dsRNA) to incite degradation of messenger RNA (mRNA) containing the same sequence as the dsRNA (Constans, 2002). Small interfering RNAs, or siRNAs are produced in at least two steps: an endogenous ribonuclease cleaves longer dsRNA into shorter, 21-23 nucleotide-long RNAs. The siRNA segments then mediate the degradation of the target mRNA (Zamore, 2001). RNAi has been used for gene function determination in a manner similar to antisense oligonucleotides (Constans, 2002). Expression vectors that continually express siRNAs in transiently and stably transfected have been engineered to express small hairpin RNAs (shRNAs), which get processed in vivo into siRNAs-like molecules capable of carrying out gene-specific silencing (Brummelkamp et al., 2002; Paddison et al., 2002). Post-transcriptional gene silencing by double-stranded RNA is discussed in further detail by Hutvágner and Zamore, 2002; Vaucheret et al., 2001; Hammond et al., 2001; Maine, 2000; Fire et al., 1998; and Timmons and Fire, 1998. Methods for using RNAi to inhibit the expression of a gene are known to those of skill in the art. See, e.g., U.S. Pat. Nos. 5,034,323; 6,326,527; 6,452,067; 6,573,099; 6,753,139; and 6,777,588. See also International Patent Publications WO 97/01952, WO 98/36083, WO 98/53083, WO 99/32619 and WO 01/75164; and U.S. Patent Publications 2003/0175965, 2003/0175783, 2003/0180945, 2004/0214330, 2005/0244858, 2005/0277610, 2007/0265220 and 2010/0058498.
Thus, it is desired to isolate tissue-specific promoters for use in controlling the expression of a gene of interest in specific tissues at particular developmental stages in Jatropha curcas other Jatropha species as well as other plant species, such as for genetic engineering of such plants. It is also desired to isolated promoters that can be used in genetic engineering of Jatropha curcas, other Jatropha species as well as other plant species. It is also desired to produce curcin-deficient jatropha plants that are non-toxic to humans and animals.